Systematic Creation of Fluorescent Fusion Polypeptides

ABSTRACT

A method for creating a plasmid for use in producing a chimeric antibody, comprising (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.

TECHNICAL FIELD

The present application is related to the field of fluorescent proteins, more specifically to systematic creation of fluorescent fusion polypeptides.

BACKGROUND

Fluorescent protein fusion has long been a method of detection of biomolecules. A fluorescent protein, derived from an aquatic creature, is fused to a protein of interest to facilitate the protein's detection by visual and photometric means. Markiv et al demonstrated that a fluorescent protein could be genetically recombined with a scFv fragment, a single antigen recognition domain from an antibody. However, Markiv's teaching could not be consistently applied to any scFv from any antibody.

There remains a need for a method to consistently, successfully recombine any scFv with any fluorescent protein, that facilitates creation of an RFAB that is consistently expressed, as a predominantly soluble product.

SUMMARY OF INVENTION

Embodiments of the present invention provide methods to consistently, successfully recombine any scFv with any fluorescent protein, that facilitates creation of an RFAB that is consistently expressed, as a predominantly soluble product.

Embodiments of the present invention provide methods to consistently manufacture functional RFABs in large quantities. The various steps described provide methods of producing RFABs that is scaled to bulk manufacture for distribution and sale.

Embodiments of the present invention provide methods of manufacturing RFABs with a 2-week turnaround time, as illustrated in FIG. 2 . Previous methods, such as shown in FIG. 1 , require a minimum of 2 weeks to 4 weeks to generate the plasmids used in the production of the RFAB protein product.

Embodiments of the present invention provide methods to generate RFABs using Gibson assembly, a method of cloning DNA sequences or gene fragments into a plasmid usable by a bacterium to produce a desired protein. This process can require only a single step, in contrast to the 6 steps required to make the original RFAB, illustrated in FIG. 1 .

Embodiments of the present invention provide methods to generate RFABs to any epitope, consistently by linker optimization. The method of Markiv et al. is hindered by sub-optimal linker sequences and un-optimized gene fragments, making the method applicable only to limited RFAB constructs. Methods provided by the present invention involve optimization of the linker sequences that enable a more stable pairing of the scFv portions of the RFAB when linked to the fluorescent protein portion. Such optimized linkers allow for the application of RFAB generation to any scFv, regardless of the class or species of the parent antibody's origin.

Embodiments of the present invention provide methods to optimize solubility by linker optimization. Apart from generation of more functional RFABs, linker optimization results in consistent solubility. More soluble RFABs are easier to extract and require less processing post-expression thus substantially reducing production time.

Embodiments of the present invention provide methods to efficiently produce RFABs, by creating scFv-containing plasmid libraries for subsequent incorporation of Fluorescent protein genes, by Gibson assembly.

An example embodiment of the present invention provides a method of creating a plasmid for use in producing a chimeric antibody, comprising: (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker having length of at least 5 amino acids; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.

An example embodiment further comprises determining the difference in length between (a) the combined length of the Fab region and the fluorescent protein, and (b) the length of the prototype Fab used by Durvasula, and, (x) if the difference is less than or equal to 5 amino acids, then modifying the Fab region by shortening the length of the Fab region by the amount the difference is less than 5 amino acids and using a linker having a length of 5 amino acids; (y) if the difference greater than 5 amino acids, then using a linker having a length equal to the difference. An example length of linker region is 15.9 Angstroms. Linker regions of length 5 to 20 amino acids can be suitable. The overall length is tailored to match that of the prototype FAB described in US2011/0268661 A1 to Markiv, Durvasula, and Kang (herein “Durvasula”, and the overall length the “Durvasula length”). U52011/0268661 A1 is incorporated herein by reference. In cases where the desired antibody/fluorescent protein is shorter than the Durvasula length, the linker can be made longer than that used in Durvasula; in cases where the desired antibody/fluorescent protein is longer than the Durvasula length, the linker can be made shorter than that used in Durvasula.

An example embodiment of the present invention provides a method of producing a chimeric antibody, comprising (a) creating a plasmid as described above, (b) inserting the plasmid into at least one of (y) chemically competent bacteria capable of protein expression from the plasmid, (z) electrocompetent bacteria capable of protein expression from the plasmid, and (c) using the bacteria to produce the chimeric antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of RFAB production as contemplated in Markiv.

FIG. 2 is an illustration of Gibson assembly.

FIG. 3 is an illustration of binding of heavy and light chains.

FIG. 4 is an illustration of a process for determining linker length.

DESCRIPTION OF INVENTION

FIG. 1 is an illustration of RFAB production as contemplated in Markiv. RFABs were first invented by Markiv et al. in 2010. They employed an older method of restriction enzyme digestion with subsequent ligation of desired DNA fragments. Briefly, the recipient plasmid and scFv gene fragment (heavy and light chain separated by a 15 amino acid linker region) are digested with restriction enzymes specific for unique DNA sequences and digested fragments are ligated together. The resulting construct is then again digested within the linker region allowing for insertion of the gene for a fluorescent protein with matching digested ends. After a correct clone is identified, it is transformed into a bacterial host that is optimized for expression of Eukaryotic proteins.

FIG. 2 is an illustration of Gibson assembly. Gibson Assembly allows the plasmid architect to bypass multiple digestion and ligation steps and bacterial propagation by performing multiple, simultaneous cloning steps with PCR templates containing overlapping regions of DNA.

FIG. 3 is an illustration of binding of heavy and light chains. The heavy and light chains are not directly bound to the fluorescent protein, in this example, mRFP. They are bound with linker regions comprising various amino acids, allowing the heavy and light regions to properly align and self-assemble. In an example embodiment of the RFAB, the linkers were of fixed length and, as the heavy and light chains of scFvs vary, linker length was not optimized to allow for proper alignment and folding of scFv regions in relation to the fluorophore in subsequent RFAB constructs. The present invention can provide methods that optimize linker length based on scFv fragment lengths for generation of new RFABs.

For every new RFAB, development of two linker regions can be required: a first linker linking the variable heavy chain fragment to the 5′ end of the fluorophore and a second linker linking the variable light chain fragment to the 3′ end of the fluorophore. The linker region can be important because enough space needs to exist between the fluorophore and each of the scFv fragments to allow for correct folding and alignment to occur. The linker region from example embodiments of the present invention involves use of a standard linker described in the literature consisting of four glycine residues and a single serine residue (GGGGS). In general for RFABs, this is the minimum linker that is used.

FIG. 4 is an illustration of a process for determining linker length. Minimum antigen binding domain regions within a FAB or full size antibody are determined through sequence analysis. These sequences are then aligned to a template RFAB, a current product that is completely soluble from a bacterial expression system. Upon alignment, the heavy and light variable regions are assessed for length as compared to the template. If the template is longer than the new RFAB, amino acids for an additional linker sequence (GnS where n=1 to 4) can be added to compensate for the difference. If the template is shorter, additional linker sequences of G4S can be added to both ends of the fluorophore and amino acids can then be removed to compensate for the length difference. In this manner, a minimum linker of G4S is always in place. Once the sequence has been determined, the RFAB can be moved to production as described in FIG. 2 .

The following references, each of which is incorporated herein by reference, can facilitate understanding of the present invention.

-   Markiv et al, Expression of recombinant multi-coloured fluorescent     antibodies in gor−/trxB E. coli cytoplasm BMC Biotechnology 2011,     11:117 doi:10.1186/1472-6750-11-117. -   Markiv et al, Module based antibody engineering: A novel synthetic     REDantibody, Journal of Immunological Methods 364 (2011) 40-49. -   Gibson D G, Young L, Chuang R Y, Venter J C, Hutchison C A 3rd,     Smith H O (2009). “Enzymatic assembly of DNA molecules up to several     hundred kilobases”. Nature Methods. 6 (5): 343-345.     doi:10.1038/nmeth.1318. PMID 19363495. -   Gibson D G. (2011). “Enzymatic assembly of overlapping DNA     fragments”. Methods in Enzymology. 498: 349-361.     doi:10.1016/13978-0-12-385120-8.00015-2. PMID 21601685. -   Wang S, Zheng C, Liu Y, Zheng H, Wang Z (2008). “Construction of     multiform scFv antibodies using linker peptide”. Journal of Genetics     and Genomics. May; 35(5):313-6. doi: 10.1016/C1673-8527(08)60045-4. -   Bird R E, Hardman K D, Jocabson J W, Johnson S, Kaufman B M, Lee S     M, Lee T, Pope S H, Riordan G S, Whitlow M. (1988). “Single-chain     antigen-binding proteins”. Science 242(2877):423-426. -   Huston J S, Levinson D, Mudgett-Hunter M, Tai M S, Novotny J,     Margolies M N, Ridge R J, Bruccoleri R E, Haber E, Crea R, et al.     (1988). “Protein engineering of antibody binding sites: recovery of     specific activity in an anti-digoxin shingle-chain Fv analogue     produced in Escherichia coli.” Proceedings of the National Academy     of Sciences. 85(16):5879-5883. -   Chappel J A, He M, Kang A S (1998). “Modulation of antibody display     on M13 filamentous phage”. Journal of Immunological Methods.     221(1-2):25-34. -   Chappel J A, Rogers W O, Hoffman S L, Kang A S (2004). “Molecular     dissection of the human antibody response to the structural repeat     epitope of Plasmodium falciparum sporozoite from a protected donor.”     Malar Journal. 3:28. -   Gu X, Jia X, Feng J, Shen B, Huang Y, Geng S, Sun Y, Wang Y, Li Y,     Long M (2010). “Molecular modeling and affinity determination of     scFv antibody: proper linker peptide enhances its activity”. Annals     of Biomedical Engineering. February; 38(2):537-49. doi:     10.1007/s10439-009-9810-2.

The present invention has been described in connection with various example embodiments. It will be understood that the above description is merely illustrative of the applications of the principles of the present invention, the scope of which is to be determined by the claims viewed in light of the specification. Other variants and modifications of the invention will be apparent to those skilled in the art.

The sequence listing in the XML file named “sequence-listing-sa152-52301 Final Export.xml”, created 2023-09-11, size 6K bytes, is incorporated herein by reference. 

We claim:
 1. A method of creating a plasmid for use in producing a chimeric antibody, comprising: (a) receiving a FAB region of the antibody; (b) receiving a fluorescent protein; (c) receiving a linker comprising at least four glycine residues and one serine residue; (d) using the Gibson assembly process to join the FAB region, the fluorescent protein, and the linker into an expression plasmid.
 2. The method of claim 1, further comprising using PCR to produce volumes of the FAB region, the fluorescent protein, and the linker for use in the Gibson assembly process.
 3. The method of claim 1, wherein receiving a FAB region comprises determining if the combined length of the FAB region, the fluorescent protein, and the linker will exceed the Durvasula length, and, if so, modifying the FAB region such that the combined length will not exceed the Durvasula length.
 4. The method of claim 1, wherein receiving a linker comprises determining the amount the length of (a) the combined length of the FAB region and the fluorescent protein, is less than (b) the Durvasula length, and receiving a linker whose length equals the determined amount.
 5. The method of claim 1, further comprising determining the difference in length between (a) the combined length of the FAB region and the fluorescent protein, and (b) the Durvasula length, and, (x) if the difference is less than or equal to 5 amino acids, then modifying the FAB region by shortening the length of the FAB region by the amount the difference is less than 5 amino acids and using a linker having a length of 5 amino acids; (y) if the difference greater than 5 amino acids, then using a linker having a length equal to the difference.
 6. A method of producing a chimeric antibody, comprising (a) creating a plasmid according to claim 1, (b) inserting the plasmid into at least one of (y) chemically competent bacteria capable of protein expression from the plasmid, (z) electrocompetent bacteria capable of protein expression from the plasmid, and (c) using the bacteria to produce the chimeric antibody.
 7. The method of claim 6 wherein step (b) comprises inserting the plasmid into chemically competent bacteria capable of protein expression from the plasmid.
 8. The method of claim 6 wherein step (b) comprises inserting the plasmid into electrocompetent bacteria capable of protein expression from the plasmid.
 9. The method of claim 6 wherein the bacteria comprises E. coli.
 10. The method of claim 6, wherein the plasmid is produced according to the method of claim
 5. 11. The method of claim 1, wherein the linker comprises at least one natural amino acid.
 12. The method of claim 1, wherein the linker comprises at least one synthetic amino acid.
 13. The method of claim 1, wherein the linker comprises at least one natural amino acid and at least one synthetic amino acid. 